AHR活化介导的ROS诱导NSCLC细胞吉非替尼耐药的作用机制

Mechanism of activated aryl hydrocarbon receptor-mediated reactive oxygen species induced gefitinib resistance in non-small cell lung cancer

目的:探讨芳香烃受体(AHR)介导的活性氧簇(ROS)诱导非小细胞肺癌(NSCLC)吉非替尼耐药的作用机制。方法:以正常支气管肺泡上皮细胞BEAS-2B为对照,Western blot检测非小细胞肺癌A549、PC-9、H1299和H1975细胞AHR的蛋白表达。AHR激动剂FICZ、吉非替尼、N-乙酰半胱氨酸(NAC)分别处理A549和PC-9细胞,MTT、激光共聚焦显微镜、流式细胞术及Western blot分别检测吉非替尼敏感性、细胞内ROS及表皮生长因子受体(EGFR)信号。结果:MTT检测显示FICZ通过上调半数最大抑制浓度(IC 50)诱导PC-9及A549细胞对吉非替尼耐药,Western blot显示FICZ可导致PC-9及A549细胞EGFR、AKT磷酸化,而NAC可遏制A549细胞中的EGFR磷酸化并逆转吉非替尼耐药。共聚焦显微镜和流式细胞仪显示,FICZ诱导的AHR活化导致PC-9及A549细胞内ROS产生增加,并且可被NAC抑制。结论:活化的AHR可通过促进NSCLC细胞中的ROS产生和EGFR信号转导介导吉非替尼耐药,此过程可被NAC抑制。

Objective:To investigate the mechanism of aryl hydrocarbon receptor(AHR)-mediated reactive oxygen species(ROS)-induced gefitinib resistance in non-small cell lung cancer.Methods:The normal bronchoalveolar epithelial cells BEAS-2 B were used as control.AHR expression in non-small cell lung cancer A549,PC-9,H1299 and H1975 cells was determined by Western blot.PC-9 and A549 cells were treated with AHR agonist FICZ and gefitinib with or without N-acetylcysteine(NAC).Gefitinib sensitivity,reactive oxygen species(ROS) levels,and EGFR signaling were detected in vitro by MTT assays,laser scanning confocal microscope,flow cytometry and Western blot,respectively.Results:MTT assay showed that FICZ induced gefitinib resistance in PC-9 and A549 cells by up-regulating the half-maximal inhibitory concentration(IC50).Western blot showed that FICZ can cause EGFR,AKT phosphorylation in PC-9 and A549 cells,while NAC can ameliorate gefitinib resistance and repress EGFR phosphorylation in A549 cells simultaneously.Confocal microscopy and flow cytometry showed that ROS production increased after AHR activation in PC-9 and A549 cells and was inhibited by NAC.Conclusion:AHR activation induces gefitinib resistance by promoting ROS production and EGFR signaling in non-small cell lung cancer,which could be reversed by NAC.